Method and Kit for Preservation of Adipose Tissue Grafts

ABSTRACT

Methods, devices, and kits are provided for use in washing or cryopreserving live cells or tissue, such as fat graft tissue.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional PatentApplication Ser. No. 62/553,322, filed Sep. 1, 2017, which isincorporated herein by reference in its entirety.

Soft tissue deformities and volume/contour deformities aftercraniofacial trauma, craniofacial deformities, congenital anomalies, anddeformities related to cancer treatment are difficult to correct.Injectable hyaluronic acid fillers are expensive, can only fill smallvolumes, and degrade with time, necessitating repeat treatments.Silicone and polyethylene implant materials are permanent foreign bodiesand have risk of long-term infection and displacement. Major soft tissueflap procedures are complex and have a high morbidity, and have asignificant donor site deformity and risk of complications at the donorsite. Autologous fat transfer is evolving as an effective means oftreating soft tissue deformities with minimal donor site morbidity andfast recovery. Fat grafting also has widespread use in cosmetic surgery.Our clinical trials strongly support this therapy, especially forcraniofacial fat grafting. Autologous fat grafting is a widely usedprocedure in plastic and reconstructive surgery, with nearly 80,000procedures performed in the United States in 2016 by plastic surgeons(American Society of Plastic Surgeons Data,https://www.plasticsurgery.org/news/plastic-surgery-statistics).

A significant difficulty with fat grafting is that approximately 63% ofthe graft volume heals and persists long term, meaning that optimalresults are obtained when at least two treatments are performed.Although the fat harvest is minimally invasive, there is time, cost, andrecovery inherent to the procedure. Moreover, the harvest usuallyrequires an operating room setting. Cost-effective devices and methodsare required to minimalize patient discomfort and recovery time.

SUMMARY

According to one aspect of the invention, a cell or tissue isolation,purification, and storage device is provided. The device comprises: avessel having a first end and a second end defining an internal chamberdivided by a filter that retains fat cells or tissue and having a volumebetween the second end and the filter of at least 3 mL; a first adaptoror connector, such as a Luer lock or slip connector, at the first end ofthe vessel defining a first opening; and a second adaptor or connector,such as a Luer lock or slip connector, at the second end of the vesseland defining a second opening.

According to another aspect of the invention, kit is provided for use inisolating and storing cells, such as a fat graft. The kit comprises:from two to 25 cell or tissue storage devices, each storage devicecomprising a vessel having a first end and a second end defining aninternal chamber divided by a filter that retains fat cells or tissueand having a volume between the second end and the filter of at least 3mL; a first adaptor or connector, such as a Luer lock or slip connector,at the first end of the vessel defining a first opening; and a secondadaptor or connector, such as a Luer lock or slip connector, at thesecond end of the vessel and defining a second opening; and at least onevessel, optionally having an adaptor or connector, such as a Luer lockor slip connector, comprising an amount of cryoprotectant able tocryopreserve at least 3 mL of cells or tissue, such as an adipose graft.

According to yet another aspect of the invention, method of preparing afat graft is provided. The method comprises: introducing (e.g.,injecting) fat tissue into the internal chamber of the device, accordingto any aspect above or herein, through the second opening of the device;introducing an amount of a cryoprotectant effective to cryopreserve thefat graft into the internal chamber of the device through the firstopening of the device; mixing the fat tissue with the cryoprotectant;and cooling the fat graft to a temperature below 0° C.

In another aspect of the invention, a method of preparing a fat graft isprovided. The method comprises: introducing (e.g., injecting) fat tissueinto the internal chamber of the device, according to any aspect aboveor herein, through the second opening of the device; introducing anamount of a wash solution effective to wash the fat graft into theinternal chamber of the device through the first opening of the device;mixing the fat tissue with the wash solution; centrifuging the devicecontaining the fat graft with the first opening on the bottom, toseparate the fat graft from the wash solution; and drawing the washsolution from the first end of the device.

In another aspect of the invention, a method of preparing a fat graft isprovided, comprising: introducing (e.g., injecting) fat tissue into theinternal chamber of the device, according to any aspect above or herein,through the second opening of the device; introducing an amount of awash solution effective to wash the fat graft or an amount of acryoprotectant effective to cryopreserve the fat graft into the internalchamber of the device through the first opening of the device; andmixing the fat tissue with the wash solution or the cryoprotectant; andeither: when wash solution is introduced, centrifuging the devicecontaining the fat graft with the first opening on the bottom, toseparate the fat graft from the wash solution; and drawing the washsolution from the first end of the device, or when cryoprotectant isintroduced, cooling the fat graft to a temperature below 0° C.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts schematically, in partial cut-away view showing thefilter, one aspect of a fat collection and storage device as describedherein.

FIG. 2 depicts schematically, in partial cut-away view showing thefilter, one aspect of a fat collection and storage device as describedherein.

FIG. 3A depicts schematically, in part, along with FIG. 3C, one aspectof a method of preserving a tissue graft, such as a fat graft asdescribed herein.

FIG. 3B depicts schematically one aspect of a storage device for use inthe methods and kits described herein.

FIG. 3C depicts schematically, in part, along with FIG. 3A, one aspectof a method of preserving a tissue graft, such as a fat graft asdescribed herein.

FIGS. 4A and 4B show results of analysis of ASCs (adipose-derived stemcells, FIG. 4A) and adipocytes (FIG. 4B) for viability using trypan bluestain, as described in Example 3.

FIGS. 5A and 5B show results of analysis of ASCs (FIG. 5A) andadipocytes (FIG. 5B) viability using trypan blue stain, as described inExample 4.

DETAILED DESCRIPTION

The use of numerical values in the various ranges specified in thisapplication, unless expressly indicated otherwise, are stated asapproximations as though the minimum and maximum values within thestated ranges are both preceded by the word “about”. In this manner,slight variations above and below the stated ranges can be used toachieve substantially the same results as values within the ranges.Also, unless indicated otherwise, the disclosure of these ranges isintended as a continuous range including every value between the minimumand maximum values.

As used herein, the terms “right”, “left”, “top”, “bottom”, andderivatives thereof shall relate to the invention as it is oriented inthe drawing figures. However, it is to be understood that the inventioncan assume various alternative orientations and, accordingly, such termsare not to be considered as limiting. Also, it is to be understood thatthe invention can assume various alternative variations and stagesequences, except where expressly specified to the contrary. It is alsoto be understood that the specific devices and processes illustrated inthe attached drawings, and described in the following specification, areexamples. Hence, specific dimensions and other physical characteristicsrelated to the embodiments disclosed herein are not to be considered aslimiting.

The terms “distal” and “proximal” refer to directions with respect tothe devices described herein, with “distal” referring to a directionaway from a user of a device, such as the direction approaching thesharp needle tip of a hypodermic syringe (the distal end of the needle),and “proximal” referring to a direction toward a user of a device, suchas the direction approaching the end of a plunger of a hypodermicsyringe in the opposite direction from the sharp needle tip, at theproximal end of the syringe. Although “distal” and “proximal”, and otherspatial relationship terms, correspond to the relative position,orientation, and/or direction of element(s) of the devices describedherein with respect to the end use of the device in typical use as apercutaneous device, or to other external reference points, thosedescriptors are provided only to describe the relative position,orientation, and/or direction of element(s) of the devices describedherein with regard to the device as a whole, and to elements thereof,and, unless otherwise indicated, do not require or infer that theelements are located, positioned, oriented, or in any physicalrelationship with an end user at any given time. Figures are schematicin nature unless otherwise identified, and are not drawn to scale, butare drawn in a manner to best depict the relationship between thevarious elements of the device drawn in the figure.

As used herein, the terms “comprising,” “comprise” or “comprised,” andvariations thereof, are meant to be open ended. The terms “a” and “an”are intended to refer to one or more.

As used herein, the “treatment” or “treating” of a condition, wound, ordefect means administration to a patient by any suitable dosage regimen,procedure and/or administration route of a composition, device orstructure with the object of achieving a desirable clinical/medicalend-point, including repair and/or replacement of a tricuspid or mitralvalve.

As used herein, the term “patient” or “subject” refers to members of theanimal kingdom including but not limited to human beings and “mammal”refers to all mammals, including, but not limited to human beings.Provided herein is a simple, cost-effective method of preserving livefat cells or tissue.

A “fluid connector” for use with syringes, cannulas, and the devices andkits described herein, can be any suitable fluid connector, alsoreferred to as a small bore fluid fitting, e.g., as are broadly-known inthe medical arts (e.g., ISO 80369). Common connectors include slip orthreaded connectors such as slip or threaded Luer connectors, as arecommon for use with medical and laboratory equipment or instruments,such as hypodermic syringes. The connectors can be matched by color orstructure to ensure proper orientation and usage of the device.

“Syringe,” as used herein refers to a medical or hypodermic syringe, asare broadly-known in the medical arts, and can be manufactured fromglass, plastic, ceramics, and/or any acceptable material. The structureand function of a syringe, and its components are broadly-known. A“cannula” is a hollow tube having a blunt end, while a “needle” forpurposes of the devices and kits described herein, refers to a hollowtub having a sharp or pointed end for ease of piercing tissue, e.g.,skin, blood vessels, or other tissue. For aspiration of fat, a cannulamay be preferred so as to lessen the chance of puncture wounds. For useof a cannula, a needle, or other “sharp” device may first be used topuncture the patient's skin. Both needles and cannulas have a distal end(for insertion into a patient), comprising an opening and, in the caseof needles, a sharp or pointed end, as are broadly-known. They also havea proximal end comprising a suitable fluid connector, such as a Luerconnector, for attachment to a syringe. Cannulas for fat harvesting caninclude a closed tip, with one or more openings in the wall of thecannula, in various patterns adjacent to its distal end. Such cannulasare broadly-available, such as Tulip CellFriendly™ cannulas andharvesting cannulas, available from Tulip Medical Products of San Diego,Calif., or from MicroAire of Charlottesville, Va., among others.

A “cryoprotectant” is a composition that can be added to live cells andprotect the cells from exposure to freezing temperatures (e.g.,cryopreservation), such as a temperature below 0° C., such as −20° C. or−80° C., resulting in substantial viability of the cells aftersubjecting the cells to freezing temperatures. The cryoprotectant istypically non-toxic when used for preservation of cells. Suitablecryoprotectants useful for protection of human cells include: dimethylsulfoxide (DMSO), human serum albumin, trehalose, and any effectivecombination of the preceding, optionally further including autologousplasma or autologous serum. Effective amounts of the cryoprotectant,such as 10% v/v DMSO, are known to those of ordinary skill, andeffective or optimized amounts of cryoprotectants are readilyascertained. Likewise, slow cooling and thawing of the fat graft may bedesirable in obtaining good viability of the harvested tissue. Referenceis made to Example 2, below for cryopreservation of fat cells.

Fat grafting is a surgical procedure that involves aspiration of fatdonor tissue from the subcutaneous tissues through a hollow borecannula. The abdomen is a common donor site. The fat graft isinjectable, making both the harvest and grafting procedure minimallyinvasive. The fat graft is composed of small particles of fat tissue,approximately 2-5 mm in diameter, that form a viscous injectable slurry.Saline solution containing epinephrine is infused into the donorsubcutaneous tissues prior to harvest. This infusion of “wettingsolution” minimizes bleeding, but also contributes an aqueous fluidcomponent to the harvested tissue that needs to be separated beforegrafting. The standard technique for harvesting and grafting, called the“Coleman Technique,” employs a hollow bore harvest cannula attached to a10 cc Luer lock syringe. The surgeon introduces the cannula through asmall incision and pulls back on the plunger to generate negativepressure. As the cannula is passed through the tissues, small particlesof fat are pulled into the aperture of the harvesting cannula, avulsedfrom the surrounding tissue with cannula movement, and drawn into thesyringe barrel. Because aqueous solution is mixed into the fat tissuefrom the wetting solution, as well as free lipids from lysed adipocytes,the fat graft material is separated from the other fractions by gentlecentrifugation in a simple table-top blood centrifuge. This device iscommonly adapted for fat grafting and present in most operating roomsfor this purpose. The concentrated fat graft can be injected into therecipient tissues from the 10 cc syringe, or transferred to smallersyringes for more precise injection.

For injection, a hollow cannula very similar to the harvest cannula isattached to the syringe barrel. All of the common fat graftinginstruments are designed to attach to Luer connectors and this fact isconsidered in the design of one aspect of the device described herein.Importantly, surgeons are used to performing the centrifuge step in theoperating room with the fat directly in the 10 cc syringe used forharvesting. The syringe plungers are simply removed and the 10 ccsyringe barrel fits into the sterilized tube of the centrifuge. Thispoint is directly relevant to the device design, because, in aspects,the device described has the same diameter and basic length of astandard, e.g., a 10 cc syringe barrel. Therefore, the fat tissuestorage device can fit into the centrifuge to facilitate post-thawwashing steps without having to change containers. The storage device ismade to work seamlessly with the current equipment and methods commonlyused by surgeons performing fat grafting.

A major deficiency of autologous fat grating is the fact that there isresorption of the fat graft during the initial healing, withapproximately 60% of the volume of the graft remaining after healing.Therefore, multiple treatments are often necessary. Since the donorharvest procedure to obtain the fat graft is a procedure that requiresan operating room setting, and special equipment to process the fattissue, it would significantly reduce cost, risk, discomfort, andimprove care if fat tissue could be stored at the time of surgery forlater use. The tissue can be stored on site in small aliquots that canbe injected into the site of injury in an office setting for refinementof the results. Because the tissue is stored on site, it would enablemultiple treatments with minimal additional cost to the original fatharvest and original fat processing.

In aspects, a device is provided in a kit comprising a cryopreservative,and special multi-function vessels that are used for mixing the fatgraft with the cryo-preservative, storage, and washing steps afterthawing. In one example, the individual vessels each hold approximately10 cc of graft material, and are stored together in a special storagecontainer that continuously records temperature and allows individuallyaliquots to be removed while preserving other vessels for continuedstorage. The large storage container that hold the individual vesselsmay be included in each kit.

In aspects, the individual single use multifunction vessels have Luerlock ports so that the system can connect with standard syringes usedfor fat grafting. A built-in filter enables infusion and drainage ofboth cryoprotectant solution and wash solution without removing the fatgraft material. The steps of adding the fat graft material, mixing withcryoprotectant, storing in a freezer, thawing, washing, and filteringall occur in the same vessel. This is a distinct advantage of thissystem. In addition to the Luer lock connectors, the device optionallycan be opened at one end to allow the fat graft to be poured intoanother sterile container as desired, or if clogging of the Luer lockport occurs. As indicated above, in one aspect, the multi-functionvessels are the same diameter as a standard 10 cc syringe and nearly thesame length, enabling them to be used in the same table-top centrifugealready employed for fat grafting and commonly found in operating rooms.

Advantages of the devices, kits and methods described herein include:

-   -   Tissue grafts can be stored on site, with very simple        preparation and at a low cost;    -   Aliquots of fat graft can be removed from storage while        remaining aliquots are left intact for future use;    -   All mixing, storage, and washing of the fat grafts occurs in the        same single use multi-function vessel;    -   The multi-function vessel is designed to work seamlessly with        commonly used fat graft equipment and standard syringes; and    -   Use of the equipment will be very intuitive for any surgeons who        perform fat grafting.

Thus, devices, kits and methods are provided herein for use inharvesting and storage of fat cells and tissue. In one aspect, a storagevessel is provided. Referring to FIG. 1, a cylindrical device 10 isdepicted. The device 10 and its components are manufactured fromsuitable materials to withstand cell and tissue cryopreservationconditions, ranging from physiological temperature (37° C.) to storageunder cryopreservation conditions, e.g., to −20° C., −80° C., or −196°C. (liquid nitrogen), such as suitable polycarbonates or polypropylenes,as are known in the cryopreservation and centrifugation arts, andsuitable metallic screens or filters. In aspects, the device 10 isdimensioned to fit into a typical clinical laboratory centrifuge able tocentrifuge devices (e.g. tubes, bottles, or syringes), that is, in oneaspect, it is dimensioned, e.g., as a typical 5 mL to 100 mL, e.g., 5mL, 10 mL, 15 mL, 20 mL, 30 mL, or 60 mL medical syringe, a typical 5 mLto 50 mL, centrifuge tube (e.g., conical centrifuge tube), or a typicalblood collection tube, that is, having an outside diameter of from 5 mmto 25 mm, e.g., from 12 mm to 25 mm, and a length (barrel length,excluding connectors) of from 70 mm to 110 mm. For example, a storagevessel for use with a 10 mL syringe may have an outside diameter rangingfrom 16.5 mm to 18 mm, and a barrel length ranging from 80 mm to 90 mm.In one example, the ratio of the length of the barrel to the outsidediameter of the barrel is at least 4:1, e.g., from 4:1 to 6:1. Inaspects, the device 10 is custom sized, but fits into a rotor, eitherwith a custom or standard rotor or a custom or standard rotor insert, ofa centrifuge, and its volume may range from 3 mL to 100 mL, or greater.A “centrifuge able to centrifuge tubes, bottles, or syringes having avolume within the range of from 1 mL to 4 L” is a centrifuge that iscommonly found in operating rooms, clinical laboratories, outpatientfacilities, and physicians offices, or a custom centrifuge and iscapable of centrifuging devices having a volume falling within the rangeof from 1 mL to 4 L, such as a 10 mL tube, but does not necessarily havethe capacity to centrifuge all volumes within that range. For example “acentrifuge able to centrifuge tubes, bottles, or syringes having avolume within the range of from 1 mL to 4 L” may only be able tocentrifuge tubes in the range of from 3 mL to 30 mL, or from 10 mL to 60mL, but not outside that range.

Referring again to FIG. 1, the device 10 comprises a vessel, depicted asa cylindrical barrel 20 that has a wall and an internal chamber (thatis, an internal void, or a lumen). The vessel is depicted as a barrel,but may have any suitable shape, though a cylindrical barrel shape maybe preferred. The device 10 also comprises a first end having a firstfluid connector 22 comprising an opening 23 extending through the firstfluid connector 22 and into the lumen of the barrel 20, and a second endhaving a second fluid connector 24 comprising an opening 25 extendingthrough the second fluid connector 24 and into to the lumen of thebarrel 20. The first fluid connector 22, the barrel 20, and the secondfluid connector 24 together form a closed fluid path between the openingof the first fluid connector 22 and the opening of the second fluidconnector 24. A filter 30 is placed within the barrel 20, adjacent tothe first fluid connector 22, and is configured to filter liquidspassing through the closed fluid path between the opening of the firstfluid connector 22 and the opening of the second fluid connector 24.That is, the filter 30 spans a complete cross-section of the lumen ofthe barrel, so that fluid passing along the closed fluid path betweenthe opening of the first fluid connector 22 and the opening of thesecond fluid connector 24, passes through the filter 30. The filter 30can be manufactured from any suitable material able to withstandcryopreservation temperatures, e.g., ranging from −196° C. to 37° C.,such as a metallic mesh, e.g., a stainless steel mesh. The filter may beany suitable pore size or mesh size, such as ranging from 50 microns (μ)to 250 μ filters, or other porous barrier that serves a filteringpurpose, such that a majority of the adipose tissue is retained by thefilter in use. The barrel 20 has an internal volume between its secondend and the filter 30 ranging from 3 mL to 100 mL, e.g., approximately 5mL, 10 mL, 15 mL, 20 mL, 25 mL, 30 mL, 50 mL, 60 mL, or 100 mL, andincrements therebetween, such as, in one non-limiting example from 9 mLto 15 mL to accommodate a tissue volume obtained using a 10 cc syringe.The device 10 optionally comprises live cells or tissue, such as livefat cells or tissue, e.g., a fat graft, such as a human fat graft,within the internal chamber of the barrel 20.

The device is designed and manufactured to withstand a G-force (amultiple of 1g, the gravitational force at the earth's surface) of atleast 500 g, e.g., at least 1000 g, 1200 g, or 1500 g, so as towithstand centrifugation conditions typical for washing and pelleting ofviable fat grafts. There are a variety of common centrifugationconditions and protocols used in preparation of fat grafts, leading toviable cells, the G-force typically ranging between 400 g and 1500 g,with typical spin durations of from 1 to 5 minutes, e.g., as shownbelow, 1200 g for three minutes.

FIG. 2 depicts a second aspect of the device 11, and is essentiallyidentical to the device 10 depicted in FIG. 1, but having a separablebarrel that separates into a first barrel portion 26, and a secondbarrel portion 28, including a slip fitting 29. The purpose of theability to separate the barrel portions 26 and 28 is to be able to openthe barrel of the device 11 to dump the contents should the devicebecome clogged. The device 11, comprises a first end having a firstfluid connector 22 comprising an opening 23 extending through the firstfluid connector 22 and into the lumen of the barrel 20, and a second endhaving a second fluid connector 24 comprising an opening 25 extendingthrough the second fluid connector 24 and into to the lumen of thebarrel formed by the first barrel portion 26 and the second barrelportion 28. The slip fitting 29 is configured to engage an inner surfaceof the first barrel portion to effectively seal the barrel and toprevent separation of the barrel portions 26 and 28, in use.Alternatively, to prevent separation of the barrel portions 26 and 28 inuse, the fitting 29 may include suitable structural elements such amating ridge on the fitting and a valley on the inside surface of thefirst barrel portion 26 such that the barrel portions 26 and 28 snaptogether. In another aspect, the fitting 29 is threaded, and the insidesurface of the first barrel portion 26 is threaded or tapped, such thatthe barrel portions screw together and can be twisted relative to eachother to separate the first barrel portion 26 from the second barrelportion 28. The fitting 29, is shown as integral to the second barrelportion 26, and alternatively, can be reversed, that is, it is integralto the first barrel portion 29.

A method for processing and storing, e.g., cryopreserving, live fatcells or tissue (a fat graft) is provided, utilizing a storage vessel asdescribed herein and exemplified by the device 10 of FIG. 1. FIGS. 3A-3Cdepict the method and elements of a kit useful for storage, e.g.,cryopreservation, of fat grafts. Referring to FIG. 3a , a fat graft 113,that is, freshly aspirated fat tissue and fat cells, are drawn into asyringe 112. The fat graft 113 is injected 140 from the syringe into thesecond end of a device 110 (the end opposite the filter), e.g.,essentially the device 10, as shown in FIG. 1, and referring to theelements as identified with respect to the device 10 of FIG. 1. Thevessel is detached from the syringe 112, and cryoprotectant 114 isinjected 142 into the first end of the device 110 (the end having thefilter). The second end of the device 110 is capped 144, and then thefirst end of the device 110 is capped 146. The device 110 is then shakento produce a mixture of the fat graft and the cryoprotectant 115. Theprocess is repeated as many times as desired, collecting, e.g., from oneto 25 tubes of fat (fat graft) from the patient and mixing the fat graftwith cryoprotectant. The fat graft is then stored 150 at a temperaturebelow 0° C., e.g., at −20° C., −80° C., or in liquid nitrogen. As usedherein, “mixing” may be by any method, including shaking, inverting,placing in a shaking water bath, gently vortexing in a vortex mixture,or any suitable method that does not substantially affect viability ofcells in the device.

FIG. 3B depicts an exemplary fat storage container 152 that can bedistributed as part of a kit for preserving fat grafts. The container152, as shown contains five filled storage devices 110 containing fatgraft mixed with cryoprotectant. Depicted are a container 152, storagevessels with individual patient labels, including indicia relating tothe patient, the container 152 also includes a safety seal, and anexternal label comprising indicia including patient information andstorage date of the fat grafts. A temperature recorder (e.g., anelectronic temperature recorder or temperature logger, as isbroadly-known) is also included to track temperature variations of thefat grafts. The container can have any shape and size adequate to holdany suitable number of storage devices 110, and is manufactured from anysuitable material able to withstand the temperature variationsassociated with the storage of the fat grafts.

Referring now to FIG. 3C, for use of the fat grafts, e.g., as a tissuefiller, the storage vessel containing the mixture 115 of the fat graftand the cryoprotectant is thawed under appropriate conditions, such ason ice, or in a refrigerator, e.g., at 4° C., and is centrifuged 154within the vessel to separate the fat graft 113′ from the cryoprotectant114′, and the cryoprotectant 114′ is then drawn 156 from the first endof the vessel. For purposes herein, the fat grafts are centrifuged atany suitable speed (RPM) for any suitable time to generate a suitableG-force for a suitable amount of time to effectively separate the fatgraft from most of the liquid the fat graft is dispersed in (e.g.,cryoprotectant or wash solutions), while retaining, optimizing, ormaximizing graft viability. The vessels are centrifuged with the firstend down because the adipose tissue will float on the cryoprotectant andwash solutions.

Wash solution 115, such as saline, water, phosphate buffered saline,serum-free medium, or any suitable wash solution, is introduced 158 intothe first end of the vessel, which is capped and shaken 160 to produce amixture 116 of wash solution and the fat graft. The vessel iscentrifuged, as described above (not shown) to separate the washsolution 115′ from the cells, and the wash solution 115′ is drawn 162from the vessel. The washing steps, 158, 160, and 162 are repeated asmany times as needed to remove cryoprotectant, free lipids, and celldebris, for example, from two to six times, e.g., three times. Aftercentrifugation, free lipids, if any, will be on top of the fat graft.The free lipids can be decanted. The vessel is flipped, and the washedfat graft 113′ is drawn 164 from the vessel into a syringe, and is readyto be injected into a patient. Optionally free lipids are removed fromthe fat graft after removal of the wash solution 115′ prior to transferof the fat graft 113″ to a syringe 164 and 166 for delivery to apatient.

Although centrifugation is particularly effective and rapid, the cellsor tissue can be separated from liquids using any useful method. Forexample and without limitation, cell or tissue material mixed with thecryoprotectant or the wash solution can be allowed to separate withoutthe use of a centrifuge, for example and without limitation, by placingand holding the device in a suitable rack for a sufficient time for thecell or tissue layer to separate from the liquid layer, and, dependingon the location of the layer or layers of separated liquid, the liquidis then drawn or decanted from the device. Where the cells or tissue area fat graft, the fat graft typically will have a lower specific gravitythan the cryoprotectant or wash solution, in which case, once separated,the higher-density liquid is drawn from the first opening of the device.Cell-free lipids, having a lower specific gravity than the fat graft,can be decanted from the second opening of the device.

Prior to mixing the fat graft with the cryoprotectant 142, afterinjection of the fat graft into the device 110, the fat graft can bewashed with a suitable wash solution, such as PBS or saline, asdescribed above (156, 158, 160, 162), and free lipids can be removed,e.g., decanted after centrifugation. After thawing and washing, the fatgraft can be further processed prior to use, such as by treating thegraft with a collagenase, filtering to remove architectural fragments,and, optionally centrifugation to separate adipocytes from othercellular elements, including the stromal vascular fraction (SVF),comprising progenitor cells, e.g., adipose-derived stem cells (ASCs),e.g., by adherence of cells of the SVF to plastic.

In one aspect, a kit is provided for use in preserving grafts, such asfat grafts. The kit comprises suitable packaging for the elements of thekit, including boxes, molded containers or inserts, suitable indiciaidentifying the contents and outlining the method of use of the elementsof the kit, as described in the method described above, and elsewhereherein. At a minimum, the kit comprises two or more storage vessels asdescribed herein, e.g., as described in reference to FIG. 1 or FIG. 2.The kit also optionally comprises one or more vessels comprising acryoprotectant as described herein, in an amount effective tocryoprotect a volume of graft, e.g., a fat graft, that can be drawn intothe vessels of the kit. For example, for two 10 mL storage vessels, foruse with a fat graft drawn into a 10 mL syringe, an amount ofcryoprotectant is provided that would be effective in cryoprotecting 20mL of fat graft material. As described above, any suitablecryoprotectant may be contained in the vessel, such as DMSO, human serumalbumin, or trehalose, or an effective combination of any of thepreceding. The cryoprotectant can be contained in one multi-use vessel,or in individual, single-use vessels that contain only enoughcryoprotectant to treat an aspirated tissue in a single vessel. The kitalso may comprise a vessel comprising a suitable wash solution in anamount appropriate for the number and size of the vessels of the kit.The vessel(s) containing the cryoprotectant or the wash solutionoptionally comprise a suitable connector, such as a Luer connector, foruse with a syringe. The kit also optionally comprises one or moresterile syringes for obtaining the fat sample from a patient orprocessing the graft as described herein. A fat harvesting cannula alsomay be included in the kit. In addition, a storage container may beincluded in the kit, e.g., as shown and described in the context of FIG.3B, and include, optionally, one or more of the following: labels forthe storage vessels, label(s) for the storage container, a temperaturelogger, or safety seals to indicate tampering with the contents of thecontainer. In one example, the kit comprises, in suitable packaging:five, 10, 15, 20, or 25 storage vessels, one or more vessel comprising acryoprotectant, one or more vessels comprising a wash solution, and astorage container including, optionally, a temperature logger.

EXAMPLE 1

To determine if the device described herein could withstand processing,and effectively separate fat from liquids, devices and methods asdepicted in FIGS. 1, 3A, and 3C, were used to process discarded freshhuman fat graft tissue collected from surgery, using 10 mL syringes, anda storage vessel essentially as depicted in FIG. 1, prepared from distalends of two 10 mL syringes solvent welded together, with a 100 μstainless steel screen placed in the first end of the device as shown.Saline (0.9% normal saline) was used both in place of a cryoprotectant,and as a wash solution. The fat graft was centrifuged at 1200g for 3minutes throughout the protocol. After the final wash, excess lipidswere decanted prior to drawing the fat graft into a syringe for transferto a patient.

EXAMPLE 2 Tissue Sample

Abdominal liposuction was performed from a 51-year-old female donor atthe Plastic Surgery Department, University of Pittsburgh. The study wasapproved by the Institutional Review Board.

Cryopreserving Solution

Dimethyl sulfoxide, DMSO (Sigma) at a concentration of 5% and 10% (V/V)in PBS was used as an intracellular cryoprotectant. To achieveextracellular cryopreservation 2% human albumin (Invitrogen) alone or incombination with 0.25M Trehalose (Sigma) was employed. Following 4combinations of cryoprotectant solutions were compared with freshnon-cryopreserved lipoaspirates.

1—10% DMSO+2%Albumin

2—10% DMSO+2%Albumin+0.25M Trehalose

3—5% DMSO+Albumin

4—5% DMSO+Albumin+0.25M Trehalose

Freezing and Thawing Procedure

Two different freezing conditions were used to cryopreserve lipoaspiratewith an object to avoid intracellular ice crystal formation: (1)Controlled freezing to −80° C. at a rate of 1° C. decrease per minute;and (2) Freezing in −80° C. freezer in Styrofoam box. Lipoaspirates werewashed twice with phosphate buffer saline, PBS (Dulbecco) bycentrifuging at 500 g for 5 minutes. Upper oil layer and lower liquidlayer is removed. Following washing, 10 ml lipoaspirate was added to a50 ml Falcon tube and equal volume of ice-cooled cryopreserving agentwas added. Controlled freeze samples were transferred to −80° C.freezer. Samples were thawed after 48 hours in a 37° C. water bath.Thawed tissue samples were washed twice with PBS to remove thecryopreserving agent.

Lipoaspirate Digestion

Fresh and thawed lipoaspirates were digested using Collagenase enzyme.Briefly, 10 ml lipoaspirates were incubated in 0.8 mg/ml type IIcollagenase (Worthington, N.J.)/3% BSA/HBSS (Sigma) for 75 minutes in ashaking water bath at 37° C. Digested solution was passed throughsterile gauze to remove the architectural fragments and centrifuged at200 g for 5 min. Upper adipocyte layer was collected in separate tubeand the stromal vascular fraction pellet was treated with ACK red bloodcells lysis buffer, passed through 70 μm cell strainer, centrifuged (200g, 5 min) and resuspended in DMEM, 10% FBS medium.

Cell Viability Analyses

The viability of adipocytes and SVF cells was analyzed using Countesscell counter (Invitrogen) following manufacturer's protocol.

Cell Culture

Stromal vesicular fraction cells were seeded at a rate of 10000 cell/cm²in 25 cm² flasks in DMEM/10%FCS/Gentamicin medium. Cells were incubatedat 37° C. in the presence of 5% CO₂. Medium was changed every 2 days andcells were counted after 8 days.

Histology Study

Lipoaspirates were fixed in 10% buffered formalin saline (FisherScientific) for 48 hours. Fixed tissue was embedded in paraffin and cutto obtain 3-5 μm sections. Hematoxylin and eosin staining was performedto visualize the architecture.

EXAMPLE 3 CRYOPRESERVATIVE STUDY

Methods: Lipo-aspirates were washed with PBS and liquid layer at thebottom and upper fat layer was removed. Equal quantity of lipo-aspirate(10 ml) was frozen under a controlled decrease in temperature or frozenat −80° C. in a Styrofoam box. Frozen aspirates were thawed after 48 hrsat 37° C. and washed with PBS. Following digestion with Collagenase,ASCs (FIG. 4A) and adipocytes (FIG. 4B) were analyzed for viabilityusing trypan blue stain. Setting the % viable cells isolated from freshfat as 100%, the percentage of viable cells following cryopreservationwas calculated and plotted. Al=Albumin, Th=Trehalose

EXAMPLE 4 CRYOPRESERVATIVE STUDY

Methods: Lipo-aspirates were washed with PBS and liquid layer at thebottom and upper fat layer was removed. Equal quantity of lipo-aspirate(7 ml) was either snap-frozen in liquid nitrogen or slow freezing wasperformed comprising, of 4 hrs at −20° C. and then at −80° C. in acryobox to lower the temperature exchange. For both freezing conditions,the following Cryopreservation agent (CPA) settings were used: 1)without CPA; 2) 2% albumin; and 3) 2% albumin+5% DMSO. Frozen aspirateswere thawed after 48 hrs at 37° C. and washed with PBS. Followingdigestion with Collagenase, ASCs (FIG. 5A) and adipocytes (FIG. 5B) wereanalyzed for viability using trypan blue stain.

The following numbered clauses describe various non-limiting aspects ofthe invention.

1. A cell or tissue isolation, purification, and storage devicecomprising: a vessel having a first end and a second end defining aninternal chamber divided by a filter that retains fat cells or tissueand having a volume between the second end and the filter of at least 3mL; a first adaptor or connector, such as a Luer lock or slip connector,at the first end of the vessel defining a first opening; and a secondadaptor or connector, such as a Luer lock or slip connector, at thesecond end of the vessel and defining a second opening.

2. The device of clause 1, wherein the device is dimensioned to fit intoa centrifuge able to centrifuge containers, e.g., tubes, bottles,syringes, or the device, having a volume within the range of from 1 mLto 4 L.

3. The device of clause 1 or 2, wherein the device is constructed towithstand centrifugation with a G-force of at least 500 g, e.g., atleast 1000 g, 1200 g, or 1500 g.

4. The device of any one of clauses 1-3, wherein the internal chamber ofthe vessel has a volume ranging from 5 mL to 100 mL, e.g., 5 mL, 10 mL,15 mL, 30 mL, or 50 mL.

5. The device of any one of clauses 1-4, dimensioned to fit into acentrifuge able to centrifuge containers, e.g., tubes, bottles, orsyringes, having a volume within the range of from 1 mL to 4 L.

6. The device of any one of clauses 1-5, wherein the filter is closer tothe second end of the vessel than the first end of the vessel.

7. The device of any one of clauses 1-6, wherein the vessel comprisestwo pieces separable pieces, forming the contiguous sealed vessel, thepieces being connected by a slip connector or a screw connector at apoint closer to the first end of the vessel than the second end of thevessel, and optionally adjacent to the first end of the vessel.

8. The device of any one of clauses 1-7, wherein the filter is a 100micron (μ) filter.

9. The device of any one of clauses 1-8, wherein the first adaptorand/or the second adapter are Luer lock adapters.

10. The device of any one of clauses 1-9, wherein the first adaptor andthe second adapter are Luer lock adapters.

11. The device of any one of clauses 1-10, having a cylindricalcross-sectional profile.

12. The device of any one of clauses 1-11, having the diameter of a 10mL medical syringe barrel.

13. The device of clause 12, the vessel having an outside diameterranging from 16.5 to 18 mm, and a length ranging from 80 mm to 90 mm.

14. The device of any one of clauses 1-11, wherein the internal volumeof the vessel between the filter and the first end of the vessel is 5 mLor greater, e.g., ranges from 8 mL to 10 mL.

15. The device of any one of clauses 1-11, the vessel having an outsidediameter ranging from 5 mm to 25 mm, and a length ranging from 70 mm to110 mm.

16. The device of any one of clauses 1-15, the vessel having a ratio ofthe length of the barrel to the outside diameter of the barrel is atleast 4:1, or from 4:1 to 6:1.

17. The device of any one of clauses 1-16, comprising live cells, suchas live fat cells, and, optionally, cryoprotectant, in the internalchamber of the vessel between the first end of the vessel and thefilter.

18. A kit for use in isolating and storing cells, such as a fat graft,comprising:

from two to 25 cell or tissue storage devices, each storage devicecomprising a vessel having a first end and a second end defining aninternal chamber divided by a filter that retains fat cells or tissueand having a volume between the second end and the filter of at least 3mL; a first adaptor or connector, such as a Luer lock or slip connector,at the first end of the vessel defining a first opening; and a secondadaptor or connector, such as a Luer lock or slip connector, at thesecond end of the vessel and defining a second opening; and

at least one vessel, optionally having an adaptor or connector, such asa Luer lock or slip connector, comprising an amount of cryoprotectantable to cryopreserve at least 3 mL of cells or tissue, such as anadipose graft.

19. The kit of clause 18, wherein the device is dimensioned to fit intoa centrifuge able to centrifuge containers, e.g., tubes, bottles,syringes, or the device, having a volume within the range of from 1 mLto 4 L.

20. The kit of clause 18 or 19, wherein the device is constructed towithstand centrifugation with a G-force of at least 500 g, e.g., atleast 1000 g, 1200 g, or 1500 g.

21. The kit of any one of clauses 18-20, wherein the internal chamber ofthe vessel has a volume ranging from 5 mL to 100 mL, e.g., 5 mL, 10 mL,15 mL, 30 mL, or 50 mL.

22. The device of any one of clauses 18-21, dimensioned to fit into acentrifuge containers, e.g., tubes, bottles, or syringes, having avolume within the range of from 1 mL to 4 L.

23. The kit of any one of clauses 18-22, further comprising a storagecontainer configured to store the two to 25 cell or tissue storagedevices, optionally including labels for labeling the storage vesselsand/or the storage container.

24. The kit of any one of clauses 18-23, further comprising atemperature logger (temperature recorder), optionally attached to thestorage container.

25. The kit of any one of clauses 18-24, wherein the filter is closer tothe second end of the vessel than the first end of one or more, or all,of the two to 25 vessels.

26. The kit of any one of clauses 18-25, wherein one or more, or all, ofthe two to 25 vessels comprises two pieces separable pieces, forming thecontiguous sealed vessel, the pieces being connected by a slip connectoror a screw connector at a point closer to the first end of the vesselthan the second end of the vessel, and optionally adjacent to the firstend of the vessel.

27. The kit of any one of clauses 18-26, wherein the filter of one ormore, or all, of the two to 25 vessels is a 100 micron (μ) filter.

28. The kit of any one of clauses 18-27, wherein the first adaptorand/or the second adapter of one or more, or all, of the two to 25vessels are Luer lock adapters.

29. The kit of any one of clauses 18-27, wherein the first adaptor andthe second adapter of one or more, or all, of the two to 25 vessels areLuer lock adapters.

30. The kit of any one of clauses 18-29, wherein one of more, or all, ofthe two to 25 vessels has a cylindrical cross-sectional profile.

31. The kit of any one of clauses 18-30, wherein one of more, or all, ofthe two to 25 vessels has a diameter of a 10 mL medical syringe barrel.

32. The kit of any one of clauses 18-31, wherein the internal volume ofone of more, or all, of the two to 25 vessels between the filter and thefirst end of the vessel is 5 mL or greater, e.g., ranges from 8 mL to 10mL.

33. The kit of any one of clauses 18-31, wherein one of more, or all, ofthe two to 25 vessels has an outside diameter ranging from 5 mm to 25mm, and a length ranging from 70 mm to 110 mm.

34. The kit of any one of clauses 18-31, wherein one of more, or all, ofthe two to 25 vessels has an outside diameter ranging from 16.5 to 18mm, and a length ranging from 80 mm to 90 mm.

35. The kit of any one of clauses 18-31, wherein one of more, or all, ofthe two to 25 vessels has a ratio of the length of the barrel to theoutside diameter of the barrel is at least 4:1, or from 4:1 to 6:1.

36. The kit of any one of clauses 18-35, wherein the cryoprotectantcomprises dimethyl sulfoxide (DMSO), human serum albumin, trehalose, ora combination of two or more of any of the preceding, such as DMSO withhSA, e.g., 5%-10% (v/v) DMSO and 2% (w/v) hSA.

37. A method of preparing a fat graft comprising: introducing (e.g.,injecting) fat tissue into the internal chamber of the device accordingto any one of clauses 1-16 through the second opening of the device;introducing an amount of a cryoprotectant effective to cryopreserve thefat graft into the internal chamber of the device through the firstopening of the device; mixing the fat tissue with the cryoprotectant;and cooling the fat graft to a temperature below 0° C.

38. The method of clause 37, wherein the device is dimensioned to fitinto a centrifuge able to centrifuge containers, e.g., tubes, bottles,syringes, or the device, having a volume within the range of from 1 mLto 4 L.

39. The method of clause 37 or 38, wherein the device is constructed towithstand centrifugation with a G-force of at least 500 g, e.g., atleast 1000 g, 1200 g, or 1500 g.

40. The method of clause 37, further comprising after cooling the fatgraft, raising the temperature of the fat graft to thaw the fat graft.

41. The method of any one of clauses 37-40, wherein the temperature ofthe fat graft during freezing or thawing is changed no more than 2° C.per minute, or no more than 1° C. per minute.

42. The method of any one of clauses 37-41, further comprisingseparating the fat graft from the cryoprotectant; drawing thecryoprotectant from the first end of the device; and washing the fatgraft by: introducing an amount of wash solution into the first end ofthe device effective to wash the fat graft, mixing the wash solutionwith the fat graft; separating the fat graft from the wash solution; anddrawing the wash solution from the first end of the device.

43. The method of any one of clauses 37-41, further comprising,centrifuging the device containing the thawed fat graft with the firstopening on the bottom to separate the fat graft from the cryoprotectant;drawing the cryoprotectant from the first end of the device; and washingthe fat graft by: introducing an amount of wash solution into the firstend of the device effective to wash the fat graft, mixing the washsolution with the fat graft; centrifuging the device containing the fatgraft with the first opening on the bottom, to separate the fat graftfrom the wash solution; and drawing the wash solution from the first endof the device.

44. The method of clause 42 or 43, wherein the fat graft is washed fromtwo to six times.

45. The method of any one of clauses 42-44, wherein the wash solution isphosphate-buffered saline or saline (e.g. 0.9% w/v normal saline).

46. The method of any one of clauses 37-45, wherein the cryoprotectantcomprises dimethyl sulfoxide (DMSO, human serum albumin (hSA),trehalose, or a combination of any of the preceding, such as DMSO withhSA, e.g., 5%-10% (v/v) DMSO and 2% (w/v) hSA.

47. A method of preparing a fat graft comprising: introducing (e.g.,injecting) fat tissue into the internal chamber of the device accordingto any one of clauses 1-15 through the second opening of the device;introducing an amount of a wash solution effective to wash the fat graftinto the internal chamber of the device through the first opening of thedevice; mixing the fat tissue with the wash solution; centrifuging thedevice containing the fat graft with the first opening on the bottom, toseparate the fat graft from the wash solution; and drawing the washsolution from the first end of the device.

48. A method of preparing a fat graft comprising: introducing (e.g.,injecting) fat tissue into the internal chamber of the device accordingto any one of clauses 1-15 through the second opening of the device;introducing an amount of a wash solution effective to wash the fat graftor an amount of a cryoprotectant effective to cryopreserve the fat graftinto the internal chamber of the device through the first opening of thedevice; and mixing the fat tissue with the wash solution or thecryoprotectant; and either: when wash solution is introduced,centrifuging the device containing the fat graft with the first openingon the bottom, to separate the fat graft from the wash solution; anddrawing the wash solution from the first end of the device, or whencryoprotectant is introduced, cooling the fat graft to a temperaturebelow 0° C.

49. The method of clause 48, wherein wash solution is introduced intothe device after the fat graft is introduced into the device; the washsolution and the fat graft are mixed; the device containing the fatgraft is centrifuged with the first opening on the bottom, to separatethe fat graft from the wash solution; and the wash solution is drawnfrom the first end of the device.

50. The method of clause 48, wherein the cryoprotectant is introducedinto the device after the fat graft is introduced into the device; thecryoprotectant and fat graft are mixed, and the fat graft is cooled to atemperature below 0° C.

The embodiments have been described with reference to various examples.Modifications and alterations will occur to others upon reading andunderstanding the foregoing examples. Accordingly, the foregoingexamples are not to be construed as limiting the disclosure.

1. A cell or tissue isolation, purification, and storage device comprising: a vessel having a first end and a second end defining an internal chamber divided by a filter that retains fat cells or tissue and having a volume between the second end and the filter of at least 3 mL; a first adaptor or connector, such as a Luer lock or slip connector, at the first end of the vessel defining a first opening; and a second adaptor or connector, such as a Luer lock or slip connector, at the second end of the vessel and defining a second opening.
 2. The device of claim 1, wherein the device is dimensioned to fit into a centrifuge able to centrifuge containers having a volume within the range of from 1 mL to 4 L and/or the device is constructed to withstand centrifugation with a G-force of at least 500 g. 3.-5. (canceled)
 6. The device of claim 1, wherein the vessel comprises two separable pieces, forming the contiguous sealed vessel, the pieces being connected by a slip connector or a screw connector at a point closer to the first end of the vessel than the second end of the vessel, and optionally adjacent to the first end of the vessel. 7.-9. (canceled)
 10. The device of claim 1, having the diameter of a 10 mL medical syringe barrel, the vessel optionally having an outside diameter ranging from 16.5 to 18 mm, and a length ranging from 80 mm to 90 mm, and optionally wherein the internal volume of the vessel between the filter and the first end of the vessel ranges from 8 mL to 10 mL.
 11. The device of claim 1, comprising live cells and, optionally, cryoprotectant, in the internal chamber of the vessel between the first end of the vessel and the filter.
 12. A kit for use in isolating and storing cells, such as a fat graft, comprising: from two to 25 cell or tissue storage devices, each storage device comprising a vessel having a first end and a second end defining an internal chamber divided by a filter that retains fat cells or tissue and having a volume between the second end and the filter of at least 3 mL; a first adaptor or connector, such as a Luer lock or slip connector, at the first end of the vessel defining a first opening; and a second adaptor or connector, such as a Luer lock or slip connector, at the second end of the vessel and defining a second opening; and at least one vessel, optionally having an adaptor or connector, such as a Luer lock or slip connector, comprising an amount of cryoprotectant, such as dimethyl sulfoxide (DMSO), human seum albumin, trehalose, or a combination of two or more of any of the preceding, able to cryopreserve at least 3 mL of cells or tissue, such as an adipose graft.
 13. The kit of claim 12, wherein the device is dimensioned to fit into a centrifuge able to centrifuge containers having a volume within the range of from 1 mL to 4 L, and/or the device is constructed to withstand centrifugation with a G-force of at least 500 g.
 14. The kit of claim 12, wherein the internal chamber of the vessel has a volume ranging from 5 mL to 100 mL.
 15. The kit of claim 12, further comprising a storage container configured to store the two to 25 cell or tissue storage devices, optionally including labels for labeling the storage vessels and/or the storage container.
 16. The kit of claim 12, further comprising a temperature logger.
 17. The kit of claim 12, wherein the filter is closer to the second end of the vessel than the first end of one or more, or all, of the two to 25 vessels.
 18. The kit of claim 12, wherein one or more, or all, of the two to 25 vessels comprises two separable pieces, forming the contiguous sealed vessel, the pieces being connected by a slip connector or a screw connector at a point closer to the first end of the vessel than the second end of the vessel, and optionally adjacent to the first end of the vessel. 19.-21. (canceled)
 22. The kit of claim 12, wherein one or more, or all, of the two to 25 vessels has a diameter of a 10 mL medical syringe barrel, the internal volume of one or more, or all, of the two to 25 vessels between the filter and the first end of the vessel ranges from 8 mL to 10 mL, one or more, or all, of the two to 25 vessels has an outside diameter ranging from 5 mm to 25 mm, and a length ranging from 70 mm to 110 mm, one or more, or all, of the two to 25 vessels has an outside diameter ranging from 16.5 to 18 mm, and a length ranging from 80 mm to 90 mm, and/or one or more, or all, of the two to 25 vessels has a ratio of the length of the barrel to the outside diameter of the barrel is at least 4:1, or from 4:1 to 6:1.
 23. (canceled)
 24. A method of preparing a fat graft comprising: introducing fat tissue into the internal chamber of the device according to claim 1 through the second opening of the device; introducing an amount of a wash solution effective to wash the fat graft or an amount of a cryoprotectant effective to cryopreserve the fat graft into the internal chamber of the device through the first opening of the device; and mixing the fat tissue with the wash solution or the cryoprotectant; and either: when wash solution is introduced, centrifuging the device containing the fat graft with the first opening on the bottom, to separate the fat graft from the wash solution; and drawing the wash solution from the first end of the device, or when cryoprotectant is introduced, cooling the fat graft to a temperature below 0° C.
 25. The method of claim 24, wherein wash solution is introduced into the device after the fat graft is introduced into the device; the wash solution and the fat graft are mixed; the fat graft is separated from the wash solution; and the wash solution is drawn from the first end of the device.
 26. The method of claim 24, wherein cryoprotectant is introduced into the device after the fat graft is introduced into the device; the cryoprotectant and fat graft are mixed, and the fat graft is cooled to a temperature below 0° C.
 27. The method of claim 26, wherein the cryoprotectant comprises dimethyl sulfoxide (DMSO), human serum albumin (hSA), trehalose, or a combination of any of the preceding.
 28. The method of claim 26, further comprising after cooling the fat graft, raising the temperature of the fat graft to thaw the fat graft.
 29. The method of claim 28, further comprising separating the fat graft from the cryoprotectant; drawing the cryoprotectant from the first end of the device; and washing the fat graft by: introducing an amount of wash solution into the first end of the device effective to wash the fat graft, mixing the wash solution with the fat graft; separating the fat graft from the wash solution; and drawing the wash solution from the first end of the device.
 30. The method of claim 28, further comprising, centrifuging the device containing the thawed fat graft with the first opening on the bottom to separate the fat graft from the cryoprotectant; drawing the cryoprotectant from the first end of the device; and washing the fat graft by: introducing an amount of wash solution into the first end of the device effective to wash the fat graft, mixing the wash solution with the fat graft; centrifuging the device containing the fat graft with the first opening on the bottom, to separate the fat graft from the wash solution; and drawing the wash solution from the first end of the device.
 31. The method of claim 26, wherein the temperature of the fat graft during freezing or thawing is changed no more than 2° C. per minute, or no more than 1° C. per minute. 